Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Currie, Michael J.; Davies, James S.; Scalise, Mariafrancesca; Gulati, Ashutosh; Wright, Joshua D.; Newton-Vesty, Michael C.; Abeysekera, Gayan S.; Subramanian, Ramaswamy; Wahlgren, Weixiao Y.; Friemann, Rosmarie; Allison, Jane R.; Mace, Peter D.; Griffin, Michael DW; Demeler, Borries; Wakatsuki, Soichi; Drew, David; Indiveri, Cesare; Dobson, Renwick CJ; North, Rachel A.

doi:10.7554/elife.92307

Title: Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Journal Article · 12 February 2024 · eLife

DOI: https://doi.org/10.7554/elife.92307 · OSTI ID:2403394

^[1];

^[2]; Scalise, Mariafrancesca ^[3]; Gulati, Ashutosh ^[4];

^[5]; Newton-Vesty, Michael C. ^[5]; Abeysekera, Gayan S. ^[5]; Subramanian, Ramaswamy ^[6];

^[7]; Friemann, Rosmarie ^[7]; Allison, Jane R. ^[8];

^[9]; Griffin, Michael DW ^[10]; Demeler, Borries ^[11]; Wakatsuki, Soichi ^[12];

^[4]; Indiveri, Cesare ^[13];

^[14]; North, Rachel A. ^[15]

Univ. of Canterbury, Christchurch (New Zealand); SLAC
Univ. of Canterbury, Christchurch (New Zealand); Stockholm Univ. (Sweden)
Univ. of Calabria, Arcavacata (Italy)
Stockholm Univ. (Sweden)
Univ. of Canterbury, Christchurch (New Zealand)
Purdue Univ., West Lafayette, IN (United States)
Univ. of Gothenburg (Sweden)
Univ. of Auckland (New Zealand)
University of Otago, Dunedin (New Zealand)
Univ. of Melbourne, VIC (Australia)
Univ. of Montana, Missoula, MT (United States); Univ. of Lethbridge, AB (Canada)
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States); Stanford Univ., CA (United States). School of Medicine
Univ. of Calabria, Arcavacata (Italy); National Research Council (CNR), Bari (Italy). Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM)
Univ. of Canterbury, Christchurch (New Zealand); Univ. of Melbourne, VIC (Australia)
Stockholm Univ. (Sweden); Univ. of Sydney, NSW (Australia)

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na⁺ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (K_D) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.

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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC); Royal Society Te Apārangi; Ministry of Business, Innovation and Employment's (MBIE)

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 2403394

Journal Information:: eLife, Journal Name: eLife Vol. 12; ISSN 2050-084X

Publisher:: eLife Sciences Publications, Ltd.Copyright Statement

Country of Publication:: United States

Language:: English

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